Peroxisomal Ubiquitin-Protein Ligases Peroxin2 and Peroxin10 Have Distinct But Synergistic Roles in Matrix Protein Import and Peroxin5 Retrotranslocation in Arabidopsis

Peroxisomal matrix proteins carry peroxisomal targeting signals (PTSs), PTS1 or PTS2, and are imported into the organelle with the assistance of peroxin (PEX) proteins. From a microscopy-based screen to identify Arabidopsis (Arabidopsis thaliana) mutants defective in matrix protein degradation, we isolated unique mutations in PEX2 and PEX10, which encode ubiquitin-protein ligases anchored in the peroxisomal membrane. In yeast (Saccharomyces cerevisiae), PEX2, PEX10, and a third ligase, PEX12, ubiquitinate a peroxisome matrix protein receptor, PEX5, allowing the PEX1 and PEX6 ATP-hydrolyzing enzymes to retrotranslocate PEX5 out of the membrane after cargo delivery. We found that the pex2-1 and pex10-2 Arabidopsis mutants exhibited defects in peroxisomal physiology and matrix protein import. Moreover, the pex2-1 pex10-2 double mutant exhibited severely impaired growth and synergistic physiological defects, suggesting that PEX2 and PEX10 function cooperatively in the wild type. The pex2-1 lesion restored the unusually low PEX5 levels in the pex6-1 mutant, implicating PEX2 in PEX5 degradation when retrotranslocation is impaired. PEX5 overexpression altered pex10-2 but not pex2-1 defects, suggesting that PEX10 facilitates PEX5 retrotranslocation from the peroxisomal membrane. Although the pex2-1 pex10-2 double mutant displayed severe import defects of both PTS1 and PTS2 proteins into peroxisomes, both pex2-1 and pex10-2 single mutants exhibited clear import defects of PTS1 proteins but apparently normal PTS2 import. A similar PTS1-specific pattern was observed in the pex4-1 ubiquitin-conjugating enzyme mutant. Our results indicate that Arabidopsis PEX2 and PEX10 cooperate to support import of matrix proteins into plant peroxisomes and suggest that some PTS2 import can still occur when PEX5 retrotranslocation is slowed.Peroxisomes are dynamic organelles housing critical oxidative metabolic reactions while sequestering harmful reactive oxygen species from the rest of the cell. In Arabidopsis (Arabidopsis thaliana), these single membrane-bound organelles are the sole site of β-oxidation and are essential for normal development (for review, see Hu et al., 2012). Triacylglycerols stored in seeds are cleaved by lipases during germination, and the released fatty acids are β-oxidized in peroxisomes to provide energy for early seedling development (for review, see Graham, 2008). Similarly, an auxin precursor, indole-3-butyric acid (IBA), is β-oxidized to active indole-3-acetic acid (IAA) in peroxisomes (Zolman et al., 2000, 2007, 2008; Strader et al., 2010; Strader and Bartel, 2011; Strader et al., 2011). IBA application enhances rooting in many plants (Woodward and Bartel, 2005a), and IAA produced from endogenous IBA promotes hypocotyl elongation, cotyledon expansion, root hair elongation, and lateral root proliferation in Arabidopsis seedlings (Zolman et al., 2001; Strader et al., 2010, 2011; De Rybel et al., 2012).The enzymes needed for β-oxidation and other peroxisomal processes are imported into the peroxisome matrix from their site of synthesis in the cytosol using proteins known as peroxin (PEX) proteins. PEX5 and PEX7 are receptors that recognize peroxisomal targeting signals (PTSs) on proteins destined for the peroxisome matrix. PEX5 recognizes a three-amino acid C-terminal PTS1 (Keller et al., 1987), and PEX7 recognizes a nine-amino acid PTS2 located near the N terminus of the protein (Osumi et al., 1991; Swinkels et al., 1991). In plants, DEG15, a peroxisomal protease, cleaves the N-terminal PTS2 region after the protein enters the peroxisome (Helm et al., 2007; Schuhmann et al., 2008). Cargo-bound PEX5 and PEX7 associate with PEX13 and PEX14 on the peroxisomal membrane (for review, see Azevedo and Schliebs, 2006; Williams and Distel, 2006) and release their cargo into the peroxisome (Fig. 1A). In yeast (Saccharomyces cerevisiae), membrane-associated PEX5 is ubiquitinated, recognized by a complex of ATP-hydrolyzing enzymes comprised of PEX1 and PEX6, and retrotranslocated out of the peroxisome to be used in additional rounds of import (for review, see Fujiki et al., 2012; Grimm et al., 2012).Open in a separate windowFigure 1.Recombination mapping of pfl36 and pfl81 reveals mutations in PEX2 and PEX10. A, PEX proteins (numbered) implicated in matrix protein import serve as receptors (PEX5 and PEX7) recognizing PTS1 or PTS2 cargo proteins, dock receptors at the peroxisomal membrane (PEX13 and PEX14), or assist in PEX5 retrotranslocation (for review, see Hu et al., 2012). In yeast, the RING-finger proteins PEX2, PEX10, and PEX12 participate as heterooligomers in different modes of PEX5 ubiquitination (Ub; for review, see Platta et al., 2013). B, GFP-ICL fluorescence is detected in both 5- and 7-d-old pex10-2 (pfl81) seedlings carrying ICLp:GFP-ICL, whereas GFP-ICL is easily detected in 5- but not 7-d-old wild-type (Wt) ICLp:GFP-ICL seedlings. Hypocotyls of light-grown seedlings were imaged for GFP fluorescence using confocal microscopy. Bar = 20 µm. C, pfl36 was mapped to the bottom of chromosome 1 near the PEX2 gene using the phenotypes of prolonged GFP-ICL fluorescence accompanied by PMDH processing defects. The number of recombinants over the number of chromosomes scored is indicated for each marker assayed. D, A gene diagram of PEX2 depicting exons as rectangles and introns as lines. A missense mutation in the fourth exon of PEX2 in pfl36 (pex2-1) changes Arg161 to Lys. Three other pex2 alleles are indicated: pex2-2, ted3 (Hu et al., 2002), and the transfer DNA insertion allele Salk_033081 that confers embryo lethality (Hu et al., 2002). E, The locations of the lesions in viable pex2 alleles are indicated on a diagram depicting the PEX2 protein domains, which include two predicted transmembrane domains (TMDs) and a C-terminal RING domain. F, pfl81 was mapped using the IBA resistance phenotype to an interval on the lower arm of chromosome 2 that contained the PEX10 gene. The number of recombinants over the number of chromosomes scored is indicated for each marker assayed. G, pfl81 (pex10-2) carries a PEX10 splicing mutation in the last nucleotide of intron 8. Four other reported pex10 mutants are indicated on the gene diagram: the pex10-1 transfer DNA insertion allele (Schumann et al., 2003; Sparkes et al., 2003) and three Targeting Induced Local Lesions In Genomes (TILLING) alleles: pex10-G93E, pex10-P126S, and pex10-W313* (Prestele et al., 2010). H, The locations of the lesions in the two viable pex10 alleles are indicated on a diagram depicting the PEX10 protein domains, which include two predicted TMDs and a C-terminal RING domain.Yeast PEX5 ubiquitination involves the peroxisomal membrane ubiquitin-protein ligases PEX2, PEX10, and PEX12 (for review, see Platta et al., 2013). The PEX12 ubiquitin-protein ligase monoubiquitinates PEX5 with the assistance of the ubiquitin-conjugating enzyme PEX4 (Platta et al., 2009), allowing PEX5 to be recycled back to the cytosol (Fig. 1A). When PEX4 is absent, yeast ubiquitin-conjugating enzyme4 (Ubc4) works with PEX2 to polyubiquitinate PEX5, marking PEX5 for proteasomal degradation (for review, see Thoms and Erdmann, 2006; Platta et al., 2007, 2013). In yeast, the Really Interesting New Gene (RING) domain of PEX10 binds both PEX2 and PEX12 RING domains to form a trimer (El Magraoui et al., 2012). PEX10 enhances in vitro ubiquitination activity of both PEX2-Ubc4 and PEX12-PEX4 (El Magraoui et al., 2012). Similarly, mammalian PEX12 enhances the in vitro ubiquitination activity of PEX10 (Okumoto et al., 2014). These findings suggest that these RING-finger proteins might act in heteromeric pairs to polyubiquitinate or monoubiquitinate PEX5 (Fig. 1A).Arabidopsis PEX2, PEX10, and PEX12 each display zinc-dependent monoubiquitination activity in vitro (Kaur et al., 2013), but the comparative functions of the Arabidopsis RING-finger PEX proteins in PEX5 ubiquitination, recycling, and degradation have not been reported. This deficiency may, in part, reflect the fact that null alleles of the RING-finger PEX genes confer embryo lethality (Hu et al., 2002; Schumann et al., 2003; Sparkes et al., 2003; Fan et al., 2005; Prestele et al., 2010). RNA interference (RNAi) lines targeting PEX2, PEX10, or PEX12 inefficiently import matrix proteins, display the Suc dependence phenotype that typically accompanies fatty acid β-oxidation defects, and are resistant to 2,4-dichlorophenoxybutyric acid (Fan et al., 2005; Nito et al., 2007), a synthetic analog of IBA (Hayashi et al., 1998). Mutation of any one RING-finger PEX gene results in disassociation or reduced levels of the PEX2-PEX10-PEX12 complex in yeast (Hazra et al., 2002; Agne et al., 2003) and mammals (Okumoto et al., 2014). It is not known whether the defects of the Arabidopsis null and RNAi lines result directly from the loss of catalytic activity of the corresponding RING-finger protein or indirectly from destabilization of the complex and consequent loss of activity of one or both of the associated RING-finger PEX proteins.Only one mutant defective in a RING-finger PEX gene has emerged from forward genetic screens for peroxisome defects in Arabidopsis. A partial loss-of-function pex12 missense allele, aberrant peroxisome morphology4 (apm4), was isolated from a GFP-PTS1 mislocalization screen (Mano et al., 2006). In addition to partially cytosolic GFP-PTS1, apm4 displays a PTS2 processing defect, Suc dependence, and 2,4-dichlorophenoxybutyric acid resistance (Mano et al., 2006), suggesting that PEX12 facilitates peroxisome protein import.In addition to roles in matrix protein import suggested by analysis of RNAi lines (Nito et al., 2007), PEX2 and PEX10 may have plant-specific roles. A pex2 missense allele, ted3, was identified as a dominant suppressor of the photomorphogenic defects of the de-etiolated1 mutant and carries a mutation near the PEX2 RING-finger domain (Fig. 1, D and E; Supplemental Fig. S1; Hu et al., 2002). Moreover, PEX10 RNAi lines display pleiotropic phenotypes not commonly found in Arabidopsis pex mutants, including variegated leaves, reduced fertility (Nito et al., 2007), organ fusions, reduced cuticular wax deposition, and changes in endoplasmic reticulum structure (Kamigaki et al., 2009). Three pex10 alleles generated by TILLING have been reported (Fig. 1G; Supplemental Fig. S2): pex10-W313* truncates the RING-finger domain and is embryo lethal, pex10-G93E germinates but displays seedling lethality, and pex10-P126S displays reduced growth in both normal air and high CO₂ conditions (Prestele et al., 2010). Although GFP-PTS1 is localized in peroxisomes in the pex10-P126S mutant, Suc dependence and IBA resistance were not reported (Prestele et al., 2010).The consequences of overexpressing a mutant version of PEX10 carrying missense mutations in the RING-finger domain (ΔZn) in wild-type Arabidopsis also hint at plant-specific roles for PEX10. PEX10-ΔZn expression confers pleiotropic phenotypes, including smaller cells, serrated leaves, inefficient photorespiration, abnormal peroxisome size and shape, and reduced peroxisome-chloroplast association (Prestele et al., 2010). However, PEX10-ΔZn plants respond like the wild type to IBA and do not require Suc, suggesting that peroxisome metabolism is not dramatically impaired (Schumann et al., 2007; Prestele et al., 2010). In contrast, expressing PEX2-ΔZn in the wild type impairs GFP-PTS1 import without conferring morphological defects, and expressing PEX12-ΔZn confers no abnormal phenotypes (Prestele et al., 2010).Here, we describe the identification and characterization of two unique mutants carrying lesions in Arabidopsis RING-finger PEX genes. We isolated pex2-1 and pex10-2 in a forward genetic screen for genes promoting peroxisomal matrix protein degradation and used these mutants to explore the roles of the corresponding proteins in peroxisome function. We found that PEX2 and PEX10 have independent but related functions that together support PEX5 recycling and matrix protein import into plant peroxisomes.     

The Calcium-Dependent Protein Kinase CPK28 Regulates Development by Inducing Growth Phase-Specific,Spatially Restricted Alterations in Jasmonic Acid Levels Independent of Defense Responses in Arabidopsis

Phytohormones play an important role in development and stress adaptations in plants, and several interacting hormonal pathways have been suggested to accomplish fine-tuning of stress responses at the expense of growth. This work describes the role played by the CALCIUM-DEPENDENT PROTEIN KINASE CPK28 in balancing phytohormone-mediated development in Arabidopsis thaliana, specifically during generative growth. cpk28 mutants exhibit growth reduction solely as adult plants, coinciding with altered balance of the phytohormones jasmonic acid (JA) and gibberellic acid (GA). JA-dependent gene expression and the levels of several JA metabolites were elevated in a growth phase-dependent manner in cpk28, and accumulation of JA metabolites was confined locally to the central rosette tissue. No elevated resistance toward herbivores or necrotrophic pathogens was detected for cpk28 plants, either on the whole-plant level or specifically within the tissue displaying elevated JA levels. Abolishment of JA biosynthesis or JA signaling led to a full reversion of the cpk28 growth phenotype, while modification of GA signaling did not. Our data identify CPK28 as a growth phase-dependent key negative regulator of distinct processes: While in seedlings, CPK28 regulates reactive oxygen species-mediated defense signaling; in adult plants, CPK28 confers developmental processes by the tissue-specific balance of JA and GA without affecting JA-mediated defense responses.     

The Arabidopsis DELLA RGA-LIKE3 Is a Direct Target of MYC2 and Modulates Jasmonate Signaling Responses

Gibberellins (GAs) are plant hormones involved in the regulation of plant growth in response to endogenous and environmental signals. GA promotes growth by stimulating the degradation of nuclear growth–repressing DELLA proteins. In Arabidopsis thaliana, DELLAs consist of a small family of five proteins that display distinct but also overlapping functions in repressing GA responses. This study reveals that DELLA RGA-LIKE3 (RGL3) protein is essential to fully enhance the jasmonate (JA)-mediated responses. We show that JA rapidly induces RGL3 expression in a CORONATINE INSENSITIVE1 (COI1)– and JASMONATE INSENSITIVE1 (JIN1/MYC2)–dependent manner. In addition, we demonstrate that MYC2 binds directly to RGL3 promoter. Furthermore, we show that RGL3 (like the other DELLAs) interacts with JA ZIM-domain (JAZ) proteins, key repressors of JA signaling. These findings suggest that JA/MYC2-dependent accumulation of RGL3 represses JAZ activity, which in turn enhances the expression of JA-responsive genes. Accordingly, we show that induction of primary JA-responsive genes is reduced in the rgl3-5 mutant and enhanced in transgenic lines overexpressing RGL3. Hence, RGL3 positively regulates JA-mediated resistance to the necrotroph Botrytis cinerea and susceptibility to the hemibiotroph Pseudomonas syringae. We propose that JA-mediated induction of RGL3 expression is of adaptive significance and might represent a recent functional diversification of the DELLAs.     

The Arabidopsis Ethylene/Jasmonic Acid-NRT Signaling Module Coordinates Nitrate Reallocation and the Trade-Off between Growth and Environmental Adaptation

Stresses decouple nitrate assimilation and photosynthesis through stress-initiated nitrate allocation to roots (SINAR), which is mediated by the nitrate transporters NRT1.8 and NRT1.5 and functions to promote stress tolerance. However, how SINAR communicates with the environment remains unknown. Here, we present biochemical and genetic evidence demonstrating that in Arabidopsis thaliana, ethylene (ET) and jasmonic acid (JA) affect the crosstalk between SINAR and the environment. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that ethylene response factors (ERFs), including OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59, bind to the GCC boxes in the NRT1.8 promoter region, while ETHYLENE INSENSITIVE3 (EIN3) binds to the EIN3 binding site motifs in the NRT1.5 promoter. Genetic assays showed that cadmium and sodium stresses initiated ET/JA signaling, which converged at EIN3/EIN3-Like1 (EIL1) to modulate ERF expression and hence to upregulate NRT1.8. By contrast, ET and JA signaling mediated the downregulation of NRT1.5 via EIN3/EIL1 and other, unknown component(s). SINAR enhanced stress tolerance and decreased plant growth under nonstressed conditions through the ET/JA-NRT1.5/NRT1.8 signaling module. Interestingly, when nitrate reductase was impaired, SINAR failed to affect either stress tolerance or plant growth. These data suggest that SINAR responds to environmental conditions through the ET/JA-NRT signaling module, which further modulates stress tolerance and plant growth in a nitrate reductase-dependent manner.     

Arabidopsis DELLA and JAZ Proteins Bind the WD-Repeat/bHLH/MYB Complex to Modulate Gibberellin and Jasmonate Signaling Synergy

Integration of diverse environmental and endogenous signals to coordinately regulate growth, development, and defense is essential for plants to survive in their natural habitat. The hormonal signals gibberellin (GA) and jasmonate (JA) antagonistically and synergistically regulate diverse aspects of plant growth, development, and defense. GA and JA synergistically induce initiation of trichomes, which assist seed dispersal and act as barriers to protect plants against insect attack, pathogen infection, excessive water loss, and UV irradiation. However, the molecular mechanism underlying such synergism between GA and JA signaling remains unclear. In this study, we revealed a mechanism for GA and JA signaling synergy and identified a signaling complex of the GA pathway in regulation of trichome initiation. Molecular, biochemical, and genetic evidence showed that the WD-repeat/bHLH/MYB complex acts as a direct target of DELLAs in the GA pathway and that both DELLAs and JAZs interacted with the WD-repeat/bHLH/MYB complex to mediate synergism between GA and JA signaling in regulating trichome development. GA and JA induce degradation of DELLAs and JASMONATE ZIM-domain proteins to coordinately activate the WD-repeat/bHLH/MYB complex and synergistically and mutually dependently induce trichome initiation. This study provides deep insights into the molecular mechanisms for integration of different hormonal signals to synergistically regulate plant development.     

Regulation of Jasmonate-Induced Leaf Senescence by Antagonism between bHLH Subgroup IIIe and IIId Factors in Arabidopsis

Plants initiate leaf senescence to relocate nutrients and energy from aging leaves to developing tissues or storage organs for growth, reproduction, and defense. Leaf senescence, the final stage of leaf development, is regulated by various environmental stresses, developmental cues, and endogenous hormone signals. Jasmonate (JA), a lipid-derived phytohormone essential for plant defense and plant development, serves as an important endogenous signal to activate senescence-associated gene expression and induce leaf senescence. This study revealed one of the mechanisms underlying JA-induced leaf senescence: antagonistic interactions of the bHLH subgroup IIIe factors MYC2, MYC3, and MYC4 with the bHLH subgroup IIId factors bHLH03, bHLH13, bHLH14, and bHLH17. We showed that MYC2, MYC3, and MYC4 function redundantly to activate JA-induced leaf senescence. MYC2 binds to and activates the promoter of its target gene SAG29 (SENESCENCE-ASSOCIATED GENE29) to activate JA-induced leaf senescence. Interestingly, plants have evolved an elaborate feedback regulation mechanism to modulate JA-induced leaf senescence: The bHLH subgroup IIId factors (bHLH03, bHLH13, bHLH14, and bHLH17) bind to the promoter of SAG29 and repress its expression to attenuate MYC2/MYC3/MYC4-activated JA-induced leaf senescence. The antagonistic regulation by activators and repressors would mediate JA-induced leaf senescence at proper level suitable for plant survival in fluctuating environmental conditions.     

Axial and Radial Oxylipin Transport

Jasmonates are oxygenated lipids (oxylipins) that control defense gene expression in response to cell damage in plants. How mobile are these potent mediators within tissues? Exploiting a series of 13-lipoxygenase (13-lox) mutants in Arabidopsis (Arabidopsis thaliana) that displays impaired jasmonic acid (JA) synthesis in specific cell types and using JA-inducible reporters, we mapped the extent of the transport of endogenous jasmonates across the plant vegetative growth phase. In seedlings, we found that jasmonate (or JA precursors) could translocate axially from wounded shoots to unwounded roots in a LOX2-dependent manner. Grafting experiments with the wild type and JA-deficient mutants confirmed shoot-to-root oxylipin transport. Next, we used rosettes to investigate radial cell-to-cell transport of jasmonates. After finding that the LOX6 protein localized to xylem contact cells was not wound inducible, we used the lox234 triple mutant to genetically isolate LOX6 as the only JA precursor-producing LOX in the plant. When a leaf of this mutant was wounded, the JA reporter gene was expressed in distal leaves. Leaf sectioning showed that JA reporter expression extended from contact cells throughout the vascular bundle and into extravascular cells, revealing a radial movement of jasmonates. Our results add a crucial element to a growing picture of how the distal wound response is regulated in rosettes, showing that both axial (shoot-to-root) and radial (cell-to-cell) transport of oxylipins plays a major role in the wound response. The strategies developed herein provide unique tools with which to identify intercellular jasmonate transport routes.Both animals and plants produce potently active lipid-derived mediators in response to wounding. These oxylipins (oxygenated lipid derivatives) include leukotrienes and prostaglandins in animals (Funk, 2001) and jasmonates in plants (Wasternack and Hause, 2013). Although these regulators frequently show structural similarities (many are cyclopentenone and cyclopentanone lipids), they operate through different signaling pathways often involving large protein complexes. For example, prostaglandins signal in part through G protein-coupled receptor complexes (Furuyashiki and Narumiya, 2011; Kalinski, 2012), and plant jasmonate signaling operates through the Skp/Cullin/F-box CORONATINE INSENSITIVE1 complex (Browse, 2009). Many oxylipins produced in response to tissue damage in metazoans act as paracrine signals to elicit defense responses in distal undamaged cells (Funk, 2001). Similarly, it is possible that jasmonates, including the biologically active derivative jasmonoyl-Ile (JA-Ile; Fonseca et al., 2009), might be transported from cell to cell in plants. However, to date, the majority of studies on oxylipin transport in plants have used exogenous jasmonates, and it remains unclear to what extent these compounds are transported between cells and tissues when produced endogenously.Based on the fact that jasmonic acid (JA) or methyl jasmonate treatments can affect defense gene expression at a distance to the sites of their application, JA was proposed to operate as a paracrine signal capable of being transported from cell to cell in tomato (Solanum lycopersicum) leaves (Farmer et al., 1992). Similar conclusions were drawn for JA in wild tobacco (Nicotiana sylvestris; Zhang and Baldwin, 1997). Isotope-labeling experiments using exogenous jasmonates have indicated JA/JA-Ile transport away from the site of application to distal tissues and even distal organs (Zhang and Baldwin, 1997; Thorpe et al., 2007; Sato et al., 2011). Additionally, grafting experiments in tomato were consistent with long-distance transport of JA/JA precursors (Li et al., 2002; Schilmiller and Howe, 2005), although other studies did not find evidence for JA transport from wounded leaves to distal unwounded leaves (Strassner et al., 2002). Concerning Arabidopsis (Arabidopsis thaliana), Koo et al. (2009) concluded that JA-Ile accumulation detected in leaves distal to the wound site resulted mainly from de novo synthesis in undamaged leaves rather than from the transport of JA/JA-Ile from the wound site. Recently, a transporter (GLUCOSINOLATE TRANSPORTER1) capable of importing JA-Ile (but not JA) into Xenopus laevis oocytes has been described (Saito et al., 2015), further supporting the possibility that jasmonates move between cells.In addition to the transport of jasmonates, there is much evidence consistent with other wound signaling mechanisms that lead to JA synthesis and JA-mediated defense responses at various distances from wounds. That is, wound-activated signaling pathways can be classified as those working near the damage site (i.e. local responses) and those operating distal to it (Rhodes et al., 2006; Wu et al., 2007). Both these types of wound responses can be difficult to study, because several types of events (including the transport of jasmonates) may contribute to JA signaling. However, there has been some progress in understanding long-distance signaling leading to distal wound responses. These mechanisms include electrical and potentially, hydraulic signaling (for review, see Koo and Howe, 2009; Farmer et al., 2014). Membrane hyperpolarizations have been recorded in wounded plants (Zimmermann et al., 2009); however, their relationship with JA synthesis or JA responses has not yet been reported. In Arabidopsis, wounding of adult-phase rosettes stimulates the leaf-to-leaf propagation of signals that (1) can be detected with surface electrodes as cell membrane depolarizations; (2) are propagated from leaf to leaf in a mechanism that requires several clade 3 GLUTAMATE RECEPTOR-LIKE (GLR) genes, including GLR3.3 and GLR3.6; and (3) can induce JA and JA-Ile accumulation in distal unwounded sites (Mousavi et al., 2013). However, even when electrical signals were compromised in both the wounded and distal leaves of a glr3.3 glr3.6 double mutant, JA responses were affected only in the distal leaf; local responses in the damaged leaf itself were similar to the wild type (Mousavi et al., 2013). Therefore, certain clade 3 GLRs operate in rosette-stage plants to extend the range of the wound response, and even if these genes are mutated, wounded rosette leaves still produce jasmonates. In summary, both jasmonates made near wounds and jasmonates made far from wounds in response to distal signals might be subject to transport within the plant.This study focused on the mobility of endogenous jasmonates produced in response to wounding. Here, we ask: how mobile are endogenous jasmonates generated in aboveground tissues in response to wounding? Our analysis was conducted throughout the vegetative phase and included different tissues that ranged from embryonic leaves (cotyledons) and roots to expanded rosette leaves. We investigated whether a glr3.3 glr3.6 double mutant that reduces leaf-to-leaf signal propagation in the adult phase (Mousavi et al., 2013) could also reduce cotyledon-to-root wound signaling in seedlings. Results from these electrophysiology experiments then led us to investigate whether JA (or JA precursors) can translocate from wounded cotyledons to roots. To do this, we used two approaches. One was based on mutants in 13-LIPOXYGENASEs (13-LOXs) that are necessary for an early step in the synthesis of the JA precursor oxophytodienoic acid. All four 13-LOXs in Arabidopsis (LOX2, LOX3, LOX4, and LOX6) are known to contribute to JA synthesis in vivo (Chauvin et al., 2013). First, LOX2 is responsible for the synthesis of a large pool of JA in wounded leaves (Bell et al., 1995), and it also produces precursors for the synthesis of arabidopside defense-related metabolites (Glauser et al., 2009). Second, LOX3 and LOX4 act together to produce the JA required for full male fertility (Caldelari et al., 2011). Third, LOX6 produces jasmonates in roots that are first separated from aerial tissues and then wounded (Grebner et al., 2013). We tested the impact of mutations in the different 13-LOXs on root JA signaling after cotyledon wounding. This was followed by grafting experiments between the wild type and the JA-deficient mutant allene oxide synthase (aos; Park et al., 2002) to test whether JA (or JA precursors) could translocate axially from wounded shoots into undamaged roots.In addition to its role in wounded roots (Grebner et al., 2013), LOX6 has been implicated in long-distance wound signaling in the adult-phase rosette, where it is necessary for most of the rapid distal expression of the JA-responsive gene JASMONATE ZIM-DOMAIN10 (JAZ10) when another leaf is wounded (Chauvin et al., 2013). This and the fact that the LOX6 promoter is active principally in xylem contact cells (Chauvin et al., 2013) provided us with the opportunity to investigate oxylipin transport within leaves. We confirmed the cellular localization of the LOX6 polypeptide with a LOX6-GUS fusion protein. We then used a lox234 triple mutant expressing a JAZ10 reporter to test whether jasmonates could be exported from xylem contact cells. These experiments led to unique insights into the transport of jasmonates across different leaf cell layers.